【作者】 李梅；

【导师】 刘玉梅；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2009， 博士

【摘要】 结球甘蓝(Brassica oleracea L. var. capitata L.)是世界各地普遍栽培的一种重要的十字花科芸薹属蔬菜作物,在我国蔬菜生产中占有举足轻重的地位。在甘蓝生产中,未熟抽薹现象时有发生,是春季甘蓝生产中需要解决的重要问题。选育耐未熟抽薹品种和新种质是解决这一问题的最经济有效的措施。对育种目标性状遗传体系的了解及相关基因的定位将有助于应用适当的育种方法对目标性状进行定向选择,提高育种效率,加速育种进程。本课题以耐未熟抽薹不同的甘蓝自交系及其配制的不同组合和分离群体为试材,对结球甘蓝抽薹时间和开花时间性状的遗传、生理及相关基因的分子标记定位等方面进行了一系列的研究,得到以下主要结论:1.主基因+多基因混合遗传模型分离分析结球甘蓝抽薹时间性状的结果表明,结球甘蓝抽薹时间的遗传受2对主基因的控制,同时存在多基因对主基因的修饰。2对主基因解释了总遗传变异的86%以上,解释了分离世代总表型变异的68%～88%。说明甘蓝抽薹时间性状主基因起决定作用。结球甘蓝抽薹时间性状是受多基因控制的数量性状,存在效应大的主基因,在进行耐抽薹育种中可采用单交重组或简单回交转育的方法进行。2.主基因+多基因混合遗传模型分离分析结球甘蓝开花时间性状的结果表明,结球甘蓝开花时间的遗传受1对主基因控制,同时存在多基因对主基因的修饰。主基因解释总遗传变异的83%以上,解释分离世代总表型变异的44%～58%,说明甘蓝开花时间性状主基因起决定作用。开花时间性状的遗传和抽薹时间性状的遗传基本一致,可以采用同样的育种方法进行品种选育。3.本研究采用Griffing完全双列杂交方法Ⅳ,对中国农业科学院蔬菜花卉研究所甘蓝课题组育成的6个结球甘蓝优良自交系的抽薹时间和开花时间性状进行配合力和遗传力分析。结果表明,自交系05521-2和04M9-2均具有良好的一般配合力,具有作为耐抽薹开花亲本材料的潜力;抽薹时间性状的广义遗传力和狭义遗传力分别为92.6%和50.04%;开花时间性状的广义遗传力和狭义遗传力分别为92.09%和67.28%,它们主要受加性效应基因控制,在早世代着手对结球甘蓝的抽薹时间和开花时间性状的选择是有效的。4.利用结球甘蓝品种间杂交的F1代经自交后得到的F2代为作图群体,采用SSR和AFLP等分子标记方法构建了结球甘蓝分子遗传图谱。该遗传图谱包含了13个连锁群,共有113个标记位点(其中包含81个SSR标记,31个AFLP标记和1个CAPS标记),连锁群长度为11 cM～146.6 cM,覆盖基因组长度的677.7 cM,平均图距为6.0 cM,连锁群上的标记数为3～19个。该分子标记连锁图谱可以进行数量性状的QTL初步定位研究。5.分子标记分析结球甘蓝抽薹时间性状结果表明,结球甘蓝抽薹时间通过区间作图检测到4个QTL,单个QTL解释表型变异的9.8%～18.7%,共解释表型变异的56.2%;其中2个QTL表现正向的加性效应,其余2个表现负向加性效应。复合区间作图定位到1个QTL,该QTL解释表型变异的18.7%,加性效应为5.4;并且与08c0954标记共分离,QTL定位在小于10 cM的区间。在耐抽薹品种选育中可利用该共分离的标记和两侧的标记进行标记辅助选择。6.分子标记分析结球甘蓝开花时间性状结果表明,结球甘蓝开花时间通过区间作图检测到4个QTL,单个QTL解释表型变异的14.5%～19.1%,共解释表型变异的66.2%;4个QTL均表现正向的加性效应。复合区间作图定位到1个QTL,该QTL解释表型变异的15%,加性效应为3.14;该QTL与08c0784-2标记共分离,QTL定位在0.73 cM的区间,可进一步精细定位和基因克隆。在耐抽薹品种选育中可利用该共分离的标记和两侧的标记进行标记辅助选择。7.根据对甘蓝的生长点进行花芽形态分化发育过程的观察,将甘蓝的花芽分化划分为5个阶段,并明确了不同阶段的主要特点:第1阶段:花芽未分化期(营养生长期)。茎尖生长锥圆滑,周边有叶原基;第2阶段:花芽将分化期。茎尖生长锥加宽,有突起,周边有苞片原基和叶原基;第3阶段:花芽分化期。花原基和苞片原基形成,并且出现更多的花原基和苞片原基;第4阶段:花器官分化期。萼片原基形成,并且花器官萼片、花瓣、雄蕊、雌蕊逐渐形成;第5阶段:现蕾期。花蕾肉眼可见。以上得出的甘蓝花芽分化时期的划分及变化的特点为室内鉴定甘蓝材料的抽薹开花的早晚提供了参考依据。8.采用ELISA方法对甘蓝花芽分化过程中的内源激素含量变化规律进行了研究,结果表明,内源激素(GA3、IAA、ABA和ZR)含量的降低将有利于花芽分化;GA3含量的升高促进结球甘蓝现蕾。ABA/ZR和IAA/ZR比值减少,GA3/ZR比值增加将有利于花芽分化的完成。综上所述,自交系05521-2和04M9-2可以作为耐抽薹亲本直接用于耐抽薹新品种的选育;栽培措施上可以采用施用GA3生长抑制剂控制未熟抽薹,该措施可作为生产上一个快速解决未熟抽薹的方法,但具体的生长抑制剂类型的选择和浓度的配比还需进一步研究;然而外施生长抑制剂耗费人力、物力,又破坏生态环境,选用耐抽薹品种才是经济有效的途径。本研究表明,甘蓝抽薹开花时间性状均存在主效的基因控制,可通过采用单交重组或简单回交转育的方法进行育种;此外利用定位到主效的QTL,在育种过程中进行耐未熟抽薹品种的辅助选择,可进一步提高育种效率。更多还原

【Abstract】 Cabbage (Brassica oleracea L. var. capitata L.) is an important vegetable crop of Cruciferous crops and is cultivated world-wide. It occupies an important position in vegetable production in China. In cabbage production, premature bolting occurred from time to time is the important issues that need to be addressed in the spring vegetable production. The most cost-effective solution to solve this problem is to develop bolting-resistant varieties and germplasms. Research on genetics and gene mapping helps to choose the appropriate breeding methods to select the target trait directionaly, to improve breeding efficiency and speed up the breeding process. This topic of genetics, physiology and related gene marker location on bolting time and flowering time was studied using different bolting-resistant cabbage inbred lines. Major conclusions are as follows:1. Analysis of major genes plus polygenes genetic segregation results show that the genetics of bolting time was controlled by two major gene and modified by polygenes; two major genes explained above 86% genetic variation, and 68%-88% of phenotype variation in segregation population. It showed that major genes played a decisive role of characteristics of the time of bolting. The bolting time character is a quantitative trait controlled by multiple genes. There is a most effective major gene, so the single cross or back cross can be used in breeding for premature bolting tolerance.2. Analysis of major genes plus polygenes genetic model segregation results showed that the genetics of flowering time was controlled by a pair of major genes modified by polygenes; major genes explained above 83% of genetic variation, and 44%-58% of phenotype variation in segregation population. It indicated that major genes played a decisive role in the characterization of flowering time. The flowering time and bolting time are simultaneously initiated. The same breeding technique could be used in cabbage variety development and selection.3. Six inbred lines from the cabbage breeding group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, was used in analyzing the combining ability and heritability of bolting time and flowering time by Griffing complete diallel crossingⅣ, the results showed that the general combining ability of inbred lines 05521-2 and 04M9-2 was desirable, which can be used as bolting-resistant parental materials; the broad heritability ability and the narrow heritability of bolting time and flowering time separately are 92.6%, 50.04% and 92.09%, 67.28%, and mainly affected by additive genes. So, the selection of bolting and flowering characters in cabbage were effective in early generations.4. A F2 generation population derived from F1 self-cross from crosses between cabbage varieties was used for constructing a genetic linkage map of SSR and AFLP molecular markers. The map included 13 linkage groups with 113 markers (including 81 SSR markers, 31 AFLP markers and one CAPS marker), the length of each linkage group was 11 cM-146.6 cM, It covered a total length of 677.7 cM with an average interval distance for 6.0 cM, and including 3-19 molecular markers for each linkage group. The molecular marker linkage map could be used QTL location.5. Molecular marker analysis showed four QTL controlling bolting time by IM, single QTL explained 9.8%-18.7% of phenotype variation and 56.2% in total; two QTL showed positive additive effect, the remaining two showed negative additive effect. MQM method mapped one QTL, it explained 18.7% of phenotype variation, the additive effect was 5.4; 08c0954 marker was co-segregated with QTL, QTL was located on less than 10 cM interval and reached a preliminary location. So the markers can be used for MAS of breeding for bolting tolerance.6. Molecular marker analysis of flowering time showed four QTL controlling by IM, single QTL explained 14.5%-19.1% of phenotype variation and 66.2% in total; all four QTL showed positive additive effect. MQM method could map one QTL, it explained 15% phenotype variation, the additive effect was 3.14; 08c0784-2 marker was co-segregated with QTL, QTL was located on less than 1 cM interval and could process fine mapping and gene cloning. So the markers can be used for MAS of breeding for bolting tolerance.7. The process of flower bud differentiation and development was observed based on the growth point of cabbage. Flower bud differentiation of cabbage can be divided into five stages as follows: Stage 1: Vegetative growth stage, characterized by a curved stem apex surrounded by leaf primordia at the basal part; Stage 2: Flower bud initiation, stem apex is widening and surrounded by bract primordia and leaf primordia at the basal part; Stage 3: Flower bud differentiation stage, characterized by floral primordia and bract primordia formation, and more floral and bract primodia are formed; Stage 4: Floral organ differentiation stage, characterized by sepal primordia formation and floral organs formation;Stage 5: Flower bud appearing stage, flower bud is visible without microscope. These characteristics of cabbage flower bud differentiation provide a reference for identifying bolting and flowering time of cabbage indoor.8. The process of cabbage flower bud differentiation of endogenous hormones was determined by ELISA method. The results show that endogenous hormones concentration (low concentration of ABA/ZR and IAA/ZR) was downward trend in stage 3, that helped flower bud to differentiate; higher GA3 concentration promoted bolting. Lower ABA/ZR and IAA/ZR concentration and higher GA3/ZR concentration were beneficial to the completion of flower bud differentiation.The results of this study showed that the inbred lines 05521-2 and 04M9-2 could be used as bolting-resistant varieties directly; GA3 inhibitors could be applied to control bolting in cultivation as a quick solution to prevent premature bolting in the production. However research on the type of growth inhibitor and the concentration should be further studied; the growth inhibitor cost manpower, material and brought damage to the ecological environment, the application of premature bolting varieties was an economic and effective way to solve this issue. This study showed that flowering time and bolting time of cabbage were controlled by major gene, the single cross and back cross breeding methods could be applied in the breeding for the bolting tolerance and QTL could be used as a tool to assist the breeding. It could improve the breeding efficiency.更多还原